Title: Endogenous biotin-binding proteins: an overlooked factor causing false positives in streptavidin-based protein detection
Authors: Tytgat, Hanne ×
Schoofs, Geert
Driesen, M.
Proost, Paul
Van Damme, E.
Vanderleyden, Jos
Lebeer, Sarah #
Issue Date: Jan-2015
Publisher: Blackwell Publishing
Series Title: Microbial Biotechnology vol:8 pages:164-168
Article number: DOI: 10.1111/1751-7915.12150
Abstract: Biotinylation is widely used in DNA, RNA and protein probing assays as this molecule has generally no impact on the biological activity of its substrate. During the streptavidin-based detection of glycoproteins in Lactobacillus rhamnosus GG with biotinylated lectin probes, a strong positive band of approximately 125 kDa was observed, present in different cellular fractions. This potential glycoprotein reacted heavily with concanavalin A (ConA), a lectin that specifically binds glucose and mannose residues. Surprisingly, this protein of 125 kDa could not be purified using a ConA affinity column. Edman degradation of the protein, isolated via cation and anion exchange chromatography, lead to the identification of the band as pyruvate carboxylase, an enzyme of 125 kDa that binds biotin as a cofactor. Detection using only the streptavidin conjugate resulted in more false positive signals of proteins, also in extracellular fractions, indicating biotin-associated proteins. Indeed, biotin is a known cofactor of numerous carboxylases. The potential occurence of false positive bands with biotinylated protein probes should thus be considered when using streptavidin-based detection, e.g. by developing a blot using only the streptavidin conjugate. To circumvent these false positives, alternative approaches like detection based on digoxigenin labelling can also be used.
ISSN: 1751-7907
Publication status: published
KU Leuven publication type: IT
Appears in Collections:Centre of Microbial and Plant Genetics
Laboratory of Virology and Chemotherapy (Rega Institute)
Laboratory of Molecular Immunology (Rega Institute)
× corresponding author
# (joint) last author

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